Identification of Plankton on Fish Pond of Oreochromis niloticus Infected by Viral Nervous Necrosis

  AUGUST-2016 VOLUME 03 NO. 02 http://rjls.ub.ac.id

  Identification of Plankton on Fish Pond of Oreochromis niloticus Infected by Viral Nervous Necrosis Uun Yanuhar ¹⁾ , Muhammad Musa ¹⁾ , Dyah Tri Rahayu ¹⁾ , Diana Arfiati ¹⁾ ₁₎ Laboratory of Water and Biotechnology, Faculty of Fishery and Marine Science

  University of Brawijaya, Indonesia. Email

  ABSTRACT

  The purpose of study is to determine the type of plankton found in cultivation pond of nile tilapia infected by VNN. The method is to identify the plankton in the water pond and testing the VNN that infects nile tilapia related to plankton eaten using Polymerase Chain Reaction (PCR). VNN identification results on the nile tilapia digestive tract stomach and intestines as well as plankton found in the stomach and intestines indicate

  VNN positively. Plankton found in the digestive tract of fish is division of Chlorophyta, Bacillariophyta, phylum Rotifera, and Crustacea. It indicates that nile tilapia infected by VNN both on the gastrointestinal tract organs, stomatch or intestines and plankton were found with positive VNN digestive duct, while plankton found in water pond is VNN negative. The

  VNN infection depends on the nature of specifications VNN on nile tilapia and the plankton in the nile tilapia digestive duct.

  Keywords: Oreochromis niloticus, plankton, Viral Nervous Necrosis.

  INTRODUCTION

  Plankton is microscopic organism that, in general, is floating in the water and able to swim in very weak movement, because their movement is influenced by the movement of water. Phytoplankton is microscopic plant, and low-level classifications of plants, while each type of phytoplankton undergoes physiological and behavioral reactions to changes in environmental quality either physically, chemically or biologically. Phytoplankton community acts as useful indicators of water quality (George, Nirmal Kumar, & Kumar, 2012). Zooplankton is microscopic animal and serves as the first consumer that eats phytoplankton. Zooplankton and phytoplankton is the food of most juvenile fish like nile tilapia (Orechromis niloticus) and they may serve as a vector of virus (Kitamura, Kamata, Nakano, & Suzuki, 2003); (Frada et al., 2014).

  In accordance with the plankton as natural feed in water, plankton is also used as food for fish. Oreochromis niloticus in Indonesia is one of the commoditiesin the fisheries sector. Because it is easy to breed, has a fast growth rate, and adaptable. In East Java, Nile tilapia can be found in various areas in the form of aquaculture ponds. The problem that arises is the declining quality of water, including the plankton and fish mortality. Lately, death of Nile tilapia in East Java, especially Blitar, is mostly caused by mass viral infections and VNN plagues. The role of plankton in this study whether it becomes vectors or supplier of amino acids for a VNN virus outbreak is still unreported. It is clear that unfavorable environmental conditions such as poor water quality will lead to an upsurge growth of various types of plankton as well as the spread of viral infections like VNN on Nile tilapia. In addition, VNN will easily infect fish which are stressed due to high stocking density and be affected by both cold water and warm water fishes (Scholz & Liebezeit, 2012). Therefore, the

  Yanuhar U. et. al.: Identification of Plankton on Fish Pond of .................................................................

  role of plankton in Nile tilapia cultivation pond for 3 weeks which a week observation and got the attention of authors to be studied repeated three times. further in this research.

  Water Sampling.

  VNN is classified in the Betanodavirus Water quality parameters observed are: group, and can be spread horizontally from brightness, temperature, pH, DO, CO

  2 , NO 3 ,

  infected fish or by exposure of VNN- PO

  4 , and TOM. Monitoring of water quality is

  contaminated water and other organisms such as much as 3 repetitions with a sampling as water microorganisms. Betanodavirus can interval is one week. Water sampling uses DO also be transmitted through natural food that is bottle and 600 ml bottle of mineral water. plankton (Yanong, 2013). For example, virus-

  Plankton sampling

  infected plankton such as Artemia, Tigriopus Plankton samples are taken by filtering japonicus (copepod), and Acetesinte medius as much as 25 L of pond water using

  (shrimp), or by organisms that are eaten. While plankton nets with a size of No.25. A sample vertically, betanodavirus can be transmitted via of water then is inserted into the film bottle the mains or fish seeds obtained in advance to and preserved with Lugol (3 drops). Then, it the offspring of fertilized egg (Gomez, Mori, is identified and counted its abundance, Okinaka, Nakai, & Park, 2010). relative abundance, diversity index, and the

  Nile tilapia infected with VNN shows index of the dominance of plankton are clinical symptoms of the Nile tilapia using the formula 1 to 4: (Oreochromis niloticus) found at the site of

  a) research that has similarities with clinical Abundance of plankton (Rice, Baird, Eaton, & Clesceri, 2012). symptoms of VNN-infected snapper and it is emphasizedby the advent of VNN genome with

  T x V

  an analysis of 294 bp PCR with primary VNN. N = x n ...................... (1)

  L x v x p x W

  This study will examine any plankton found in water, plankton found in the digestive tract of Where, nile tilapia, and VNN genome analysis as RNA N = phytoplankton Total Number (ind/L). viruses in the digestive tract organ of nile tilapia, plankton found in digestion tract of nile n = Total of phytoplankton in each tilapia and plankton species identified, the range of sight.

  T = Cover glass width (20 x 20 mm) . condition water and environment and role of plankton in the spread of Viral Nervous L = The Width of one range of sight Necrosis (VNN) nile tilapia cultivation pond.

  (πr2 mm2). r = radius of range of sight.

MATERIALS AND METHODS

  b) Relative Abundance of Plankton.

  Sample.

  The samples are Nile tilapia infected by = 100% ....................... (2)

  Viral Nervous Necrosis (VNN) and the water pond that obtained from fisheries farm in Where, Blitar, East Java, Indonesia. KR= Relative Abundance. ni = i number of individuals.

  Research design.

  N = Total number of individuals. The research design is observational data using a laboratory analysis. Observations of

  Yanuhar U. et. al.: Identification of Plankton on Fish Pond of .................................................................

  c)

  Polymerase Chain Reaction

  The diversity index using Shannon- Wiener Index (Ghosh & Biswas, 2015). After extraction of RNA, then the amplification process in the order of the

  ′ s

  H = − ∑ pi log pi .................... (3) PCR process revers-transcriptase is

  i =1

  performed using the Quick Access AMV® Where,

  RT-PCR (Promega) followed by nested PCR pi = the proportion of importance value using Go Taq® Green Master Mix of the i (pi = ni/N). (Promega) and using the primary pair of H' = Diversity Index.

  VNN Forward 2 (F2): AGT GTC CGT GTG Ni = the number of individual of i

  TCG CAT CT and Reverse 3 (R3): CGA GTC number. AAC GGT GAA ACG GA. N = Total Number of individuals.

  The reaction process is conducted in S = Number of species in the samples. accordance with the protocol of each kit, to a final concentration of each primary 2.5 d)

  The domination Index using Simpson’s µl. Then, enter the template of RNA, a index (D) (Ghosh & Biswas, 2015). positive control and a negative control in

  2

  the PCR machine (thermal cycler). Ensure ....................................... (4)

  D = Σ (pi) that the program to be run is VNN. The Where, setting of the temperature on a thermal D = Domination Index. cycler is as Table 1. Pi = relative Abundance.

  Table 1. The setting of the temperature on a thermal cycler. Temperature No Reaction Time Cycle Number (

  C)

  1. Incubation 45 30 min.

  1

  2. Inaktivasi Reverse-transcriptase 94 2 min.

  1

  3. Denaturation 94 30 s

  4. Annealing 60 30 s

  40

  5. Extension 72 45 s

  6. Final extension 72 10 min.

  1 Electrophoresis Agarose

  After the amplification process is complete, take 10 μL of each sample and add 2 μL of staining solution (6x loading dye) on parafilm paper, then homogenized. Furthermore, around 10 μL of the sample and marker put into agarose gel and sink slowly. Then attach the electrophoresis and turn the power supply with the power of 100 V, 400 mA for 60 minutes.

  After completion of the process of electrophoresis, remove the gel. Soak gel into the buffer which is added by SyBr save (Invitrogen) for 15 minutes. Put gel in gel documentation, then observe and document using the camera.

  Yanuhar U. et. al.: Identification of Plankton on Fish Pond of .................................................................

RESULTS AND DISCUSSION

  Water quality Quality of water in the Nile tilapia cultivation pond was shown in Table 2.

  Table 2. The result of water quality parameter measurement in the cultivation pond. No Parameter Unit Week observation Mean Standard Quality

  1 ^st

  2 ^nd

  3 ^rd

  1 Temperature C

  31

  32

  30

  31

  25

• – 32 ^(*)
• – 40 ^(*) 3 pH -
• – 8,5 ^(*)
• – 10 ^(**)

  25

  (**) Marion (1998).

  ind/L (Goldman and Horne, 1983).

  7

  to 10

  4

  phosphorous in conditions of low concentrations in water. The Nile tilapia cultivation pond is included into the water with moderate fertility levels (mesotrofik) and the fertility of water is having plankton ranged from 10

  2 and

  The result of the calculation of the abundance of plankton (Table 3) showed that phytoplankton abundance was highest in week 1 and then decreased in week 2 and increased its abundance in week 3. Overall, the highest contributor to the total value of abundance phytoplankton on each week are Cyanophyta division. Erdina et al. (2013), suggests that Cyanophyceae is able to survive in conditions without light and indicators of their high content of organic material in the water. Algae is capable of taking CO

  Abundance (N)

  Based on the calculation of the relative abundance of phytoplankton found that overall (week 1 to 3) percentage of the value of the highest relative abundance is occupied by division of Cyanophyta (genus Merismopedia), followed by the division of Bacillariophyta then division of Chlorophyta. The result of the calculation of abundance (N) in Table 2 indicates that the division of Cyanophyta has high abundance. The results of relative abundance and abundance of division of Cyanophyta (Merismopedia sp.) are high on every week due phytoplankton of the division was able to adapt to the aquatic environment and the availability of nutrients needed by the phytoplankton.

  Relative abundance

  The results of the identification of plankton on the Nile tilapia cultivation pond of observations on week 1 to 3 obtained the kinds of phytoplankton and zooplankton. Phytoplankton consists of three divisions: division of Chlorophyta (2 genus), Cyanophyta (1 genus) and Bacillariophyta (7 genus). Zooplankton consists of three phyla which are phylum Arthropods (1 genus), crustaceans (1 genus) and Rotifera (1 genus).

  Plankton identification.

  8 TOM mg/L 12,64 13,90 11,38 12,64 <50 ^(**) Notes: (*) SNI (Indonesia National Standard) (2009).

  25

  7 PO ⁴ mg/L 0,05 0,05 0,05 0,05 <0,2 ^(**)

  10 ^(**)

  6 NO ^{3 mg/L 1,14 1,09 1,13 1,12}

  1

  5 CO ^{2 mg/L 5,99 9,08 3,99 6,32}

  4 DO mg/L 6,08 6,76 7,43 6,76 > 3 ^(*)

  2 Brightness cm

  6

  7

  30

  25

  25

  8 7 6,5

• Diversity Index Value (H’).
• Domination Index Value (D).

  33

  2 Tablelaria

  4

  6

  2 Diatoma

  2

  3

  5 Subtotal

  34

  47 b. Zooplankton Crustacea Calanus

  2

  1

  2 Rotifera Keratella

  2

  1 Subtotal

  3

  1

  2 Total

  37

  34

  3

  6 Surirella

  Yanuhar U. et. al.: Identification of Plankton on Fish Pond of .................................................................

  Division/Philum Genus Total (ind/ml) Week 1 Week 2 Week 3 a. Phytoplankton Chlorophyta Netrium

  Table 3. The result of abundance, diversity and domination index. Division /Phylum Week

  1

  2

  3 N (ind/L) -Pi log ^{2 Pi D N (ind/L) -Pi log 2 Pi D N (ind/L) -Pi log 2 Pi D} Phyto-plankton Chlorophyta 23,706 0.141 0.000 59,265 0.409 0.005 47.412 0.311 0.002 Cyanophyta 758,596 0.166 0.769 379,298 0.448 0.351 568.947 0.311 0.563 Bacillariophyta 82,971 0.540 0.002 201,501 1.210 0.028 142.236 0.908 0.007 Total 865,273 0.847* 0.771** 640,064 2.067* 0.384** 758.595 1.53* 0.572** Zooplankton Crustacea

  11,853 0.526 0.111 Arthropoda 11,853 0.5 0.25 11,853 0.526 0.111 Rotifera 11,853 0.5 0.25 11,853 0.526 0.111

  Total 0* 0** 23,706 1* 0.5** 35,559 1.578* 0.333** Notes:

  Table 4. Plankton composition analysis result in digestion tract of Nile tilapia (Oreochromis niloticus). Diversity Index (H')

  Phytoplankton and zooplankton diversity index, the calculation results in Table 3, indicate that the nile tilapia cultivation pond included in the criteria low to moderate H'. According Utami (2001), the diversity index criteria are divided into three types of low diversity with H'<1, moderate diversity when 1<H'<3 and high diversity when H'>3. According to Krebs (1989), moderate diversity could mean that ecosystem is in fairly good condition, where the spread of the individual or the type of phytoplankton is almost evenly. Low diversity indicates the tendency of dominant species

  1

  1

  1

  2 Bacillariophyta Navicula

  18

  15

  22 Nitzschia

  5

  4

  8 Achnantes

  2

  49

  Yanuhar U. et. al.: Identification of Plankton on Fish Pond of .................................................................

  in an ecosystem due to the instability of environmental factors and population. The description of plankton observations with Olympus BX331 microscope is shown in Figure 1 and Figure 2.

  Figure 1. Phytoplankton.

  Calanus Polypemus Keratella Figure 2. Zooplankton.

  Domination Index (D). a structure and dominance of different types Based on Table 3 on the dominance with other water.

  index of phytoplankton and zooplankton in Analysis result of VNN-infected Nile tilapia. week 1 and 2, there is a plankton species

  The observation of the morphology and that dominates which are phytoplankton of behavior of the Nile tilapia showed the division of Cyanophyta (genus symptoms that fish infected with VNN. The

  Merismopedia) and in zooplankton, there is visible symptoms are fish swimming at the no species that dominate in the water. The surface, swim angled and loss of balance, kinds of phytoplankton have a different weak movement, eyes bulging out and response to the ratio of nutrients dissolved blackish-red, the body of mucus and body in water. This condition causes color is pale and some fish showed a phytoplankton community in one water has darkening of the color of the body as shown

  Yanuhar U. et. al.: Identification of Plankton on Fish Pond of .................................................................

  Figure 4. Amplification of VNN Genome found in Intestine Nile tilapia: (1) Marker DNA lambda 1 bp; (2) Positive control; (3) Negative control; (4) Positive sample VNN 294 bp.

  Figure 3. VNN-Infected nile tilapia morphological condition.

  OIE (2013) explains that there is a change in the habits of the VNN-infected fish like swimming round and round, upside down (whirling) or stomach position is above due to the swim bladder inflammation. Sometimes the

  VNN-

  Figure 5. Amplification of VNN Genome in the plankton found in the digestive tract of

  infected fish swim vertically to the bottom

  Nile tilapia (Gastric and Intestine): (1)

  of the pond or swimming round and round

  Marker DNA lambda 1 bp; (2) Positive

  quickly or stomping the head to the

  control; (3) Negative control; (4)

  surface of the water. VNN on Nile tilapia Positive sample VNN 294 bp. detection by PCR test on primary base of 294 bp showed that the intestine is

  According to Table 2, the water quality positively infected by VNN. conditions Nile tilapia cultivation pond is still good for the growth of plankton and aquatic

  Indication of the presence of plankton

  organisms as well as Nile tilapia itself. The and VNN in Nile tilapia. results of measurements of water quality

  VNN genome amplified by PCR on Nile parameters on week 1 to 3 are still in the tilapia and plankton is shown in Figure 4 range of threshold quality standards and 5. established for aquaculture activities.

  Yanuhar U. et. al.: Identification of Plankton on Fish Pond of .................................................................

  However, brightness value of the water is low. This is caused by nile tilapia cultivation pond is included a shallow pond with a pond depth only about 25 cm. In addition, the bottom of the pool is still visible on the surface of the water so that it can be assumed that the brightness value of 100% at a depth of 25 cm.

  The existence of VNN in water is affected by environmental conditions such as physico- chemical parameters of water. The most influencing water quality parameter on the existence of VNN is pH, temperature and TOM. The pH value, temperature and TOM range from 6-8, 30-32°C, and 11.376 to 13.904 mg/L (Table 2). VNN can survive in waters with environment pH ranging from 2-

  9. While at pH 11-12, it will cause the inactivation of the virus (Munday, 2003). At a temperature of 25°C or higher temperature, it can significantly affect VNN infection of the fish. The virus is able to survive the acidic pH conditions even at a temperature of 37°C (OIE, 2013). However, when the water temperature is more than 31°C, it will inhibit the proliferation of betanodavirus or VNN (Yuasa, Koesharyani, & Mahardika, 2007). While, the organic materials are associated with the build up of feces at the bottom of the water. If the feces is derived from the nile tilapia that were infected by VNN the more feces come out and piled up in the bottom of the pool, the more the virus found in the water and likely nile tilapia can be infected by VNN.

  Merismopedia sp. is abundant in water temperatures ranging from 27 to 30°C. While at temperatures ranging from 27 to 31°C, Merismopedia species sp. is found abundantly in the waters of the freshwater reservoir. In addition, an increasing number of individuals Cyanophyta are due to an increase in nutrients in the water. Thus, the temperature obtained during observations with a range between 31-32°C is a good temperature range for growth of Cyanophyta. Moreover, the fluctuations of the concentration of nutrients (nitrate and ortopfosfat) during the observation show the fluctuations of the number of cells in each week (Mahar, Larik, Narejo, & Jafri, 2010).

  Based on Table 3, the abundance of zooplankton in week 1 is not found, while on week 2 and week 3, the total zooplankton abundance has increased. The existence of zooplankton is affected by several factors. The biotic factors such as the availability of food, the number of predators and competition are all factors that determine species composition of zooplankton. Zooplankton abundance has increased and decreased due to the factors of each zooplankton itself, such as growth, death, vertical distribution, and different migration and a change in water quality. Table 3 is based on analysis of the composition of plankton found in the digestive tract of the stomach and intestines of nile tilapia infected by VNN which is seen each type of plankton from division of Cyanophyta and bacillariophyta, and the type that is a type of crustacean, zooplankton and rotifera.

  Based on the identification plankton of the digestive tract that Nile tilapia prefer Phytoplankton mostly of the genus of Nitzschia, Navicula, Achnantes, Tablelaria, Surirella, and Diatoma as well as a small portion of the genus Netrium. Where as zooplankton is genus of Calanus and Keratella. The plankton contains amino acids. The genus of Nitzschia, Navicula, and Achnantes contains the amino acid of aspartic acid and lysine (Scholz & Liebezeit, 2012). According the genus of Calanus and Keratella contains the amino acid of lysine and arginine (Cowey & Corner, 1963) and aspartic acid, arginine and lysine (Guisande,

  N. (2012). Study on the influence of hydro-chemical parameters on phytoplankton distribution along Tapi

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  Yanuhar U. et. al.: Identification of Plankton on Fish Pond of .................................................................

  ACKNOWLEDGEMENT

  Achnantes) and the phylum Crustacea (Calanus) and Rotifera (Keratella). Both plankton in the digestive tract of nile tilapia and VNN in gastrointestinal tract of infected nile tilapia contains the amplified VNN with a length of 294 bp.

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  CONCLUSIONS

  From the Figure 5, digestive tract of Nile tilapia infected by VNN with the target is the gastric and intestines showed positive VNN with ribbon length 294 bp. Similarly, plankton identified from the digestive tract of the type found plankton Chlorophyta (Netrium), and Bacillariophyta (Navicula, Nitzschia, and Achnantes) and the phylum Crustacea (Calanus) and Rotifera (Keratella). This shows that the amino acids contained in the host body (plankton) have compatibility amino acids required in the virus reproduction. So it can be indicated that the genus Nitzschia, Navicula, Achnantes, Keratella and Calanus can be a vector in the spread of VNN on nile tilapia body through the process of predation may also be referred to as a horizontal transmission.

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